Table 2.

Control parameters for the autologous and allogeneic finished products (i.e., in-process indirect assessments and endpoint direct assessments). AB, Alcian Blue; ACAN, aggrecan; C, conforming; COL 2, collagen II; DMMB, dimethylmethylene blue; ECM, extracellular matrix; GAG, glycosaminoglycan; h, hours; HE, hematoxylin and eosin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NC, non-conforming; PBS, phosphate-buffered saline; RT-PCR, real-time polymerase chain reaction.

Control/Assessment Type	Control Parameters	Control Methods	Targets/Acceptance Criteria	Autologous Protocol Assessment		Allogeneic Protocol Assessment
				C	NC	C	NC
1. Endpoint Direct Assessment 1 of Finished Product Lot	Chondrogenic Gene Induction in 3D	RT-PCR	Induction of COL2 and ACAN	✓	-	✓	-
	Cartilage GAG Presence (Total) in 3D	DMMB	Cartilage GAG presence ≥ 0.25 µg/mg	✓	-	✓	-
	Cartilage ECM Presence (Histology) in 3D	HE	Presence of staining	✓	-	✓	-
		ACAN	Presence of staining	✓	-	✓	-
		AB	Presence of staining	✓	-	✓	-
	Cellular Viability in 3D	MTT	Presence of MTT signal	✓	-	✓	-
	Homogeneity of Cell Presence Across the Construct	MTT signal homogeneity; Histology	Homogeneous staining of the whole surface of the construct	✓	-	✓	-
	Cells and Synthetized ECM Localized in 1 Layer of the Construct	MTT; Histology	Presence of cells and ECM in one layer of the construct (absent from the other layer)	✓	-	✓	-
	Cells Localized in Lacunae	Histology	Presence of cells in the lacunae	✓	-	✓	-
	Cellular Morphology	Histology	Rounded cellular morphology	✓	-	✓	-
	Homogeneous ECM Presence Across the Construct	Histology	Homogeneous presence of ECM across the construct	✓	-	✓	-
	Significant ECM Deposition Within the Construct	Histology	Significant ECM deposition in one layer of the construct	✓	-	✓	-
2. In-Process Indirect Assessment 2 of Finished Product Lot	Cell Viability in Monolayer Control Cultures	Cell enumeration; Operator assessment	Cellular viability ≥ 75% before control plate seeding; limited amounts of floating dead cells; induction medium consumption	✓	-	✓	-
	Cellular Adhesion in Monolayer Control Cultures	Operator assessment	Presence of ≥60% adherent cells 24 h after seeding; absence of significant cellular detachment	✓	-	✓	-
	Cellular Proliferation in Monolayer Control Cultures	Operator assessment	Appropriate proliferative cellular morphology adoption, proliferation rate, and proliferation homogeneity in monolayer	✓	-	✓	-
	Cellular Population Purity in Monolayer Control Cultures	Operator assessment	Absence of observable cell sub-population presence	✓	-	✓	-

¹ Destructive control process performed directly on the finished product, requires dedicated replicate production. ² Non-destructive control process performed on cell recovery control cultures or on manufacturing process retention samples.